O glcnac

TCR stimulated CD8⁺ T cells were purified by magnetic separation (Stemcell Technologies) prior to protein extraction. Standard immunoblotting protocols were used19 (link). Blots were probed with antibodies recognizing, dilutions and clone/catalog numbers in brackets: c-Myc (1:1000, #9402), pY694 STAT5 (1:2000, #9351), pan STAT5 (1:1000, #9363), pT389 S6K (1:1000, #9239), S6K (1:1000, #5610) (Cell Signalling Technology), SMC1 (1:5000, A300-055A, Bethyl), β–tubulin (1:5000, H-235) (Santa Cruz), OGT (1:1000, DM17, Sigma) and O-GlcNAc (1:3000, RL2, Abcam). Lysate made from 2 × 10⁸ day 6 CTLs were used for the succinylated wheat germ agglutinin (sWGA) affinity purifications. One half of the lysate was treated with CpOGA and other half was left untreated. Before sWGA affinity purifications, the lysates were precleared with agarose beads for 30 min at 4 °C. 50 µl sWGA beads (L-1020S, Vector Laboratories) were incubated for 16 h with the lysates and beads were washed 4 times with PBS before adding the LDS-loading buffer. For GFP-myc immune-purification, 2 × 10⁸ GFP-Myc^KI CTLs were lysed and half the lysate was treated with CpOGA or left untreated before the immune-purification. The lysates were incubated with GFP-binder agarose beads (ChromoTek) for 90 min at 4 °C. Beads were washed three times with NP40 buffer before adding the LDS-loading buffer for immunoblotting.